oocyte-degeneration rates and abnormal fertilization rates especially whenthe zona pellucida is particularly thick and/or hard to be penetrated andwhen the oolemma is more fragile. This laser assisted ICSI procedure couldbe the method of choice in case of fragile oocytes or when only very fewoocytes are obtained.

Wednesday, October 25, 20004:45 P.M.

O-223

Pronuclear Formation of Human Oocytes Excluded from IVF-ET Pro-gram in an Oocyte Microsurgery Program.* 1S. H. Jun,1,2J. M. Lim,1S. E. Park,1,2H. M. Chung,1,2H. Shim,1,2K. Y. Cha. 1Infertility MedicalCenter of CHA General Hospital,2College of Medicine, Pochon CHAUniversity, Seoul, Korea.

Objective: We have attempted to develop a pronuclear transfer system forrescuing unfertilized oocytes (UFOs) and developmentally incompetentoocytes (IOs) that excluded from IVF-ET program. This study was under-taken to establish an effective method for pronucleus (PN) formation ofUFOs and IOs in the system.

Design: Randomized, prospective study using human oocytes retrievedfrom stimulated cycle.

Materials and Methods: Oocytes were retrieved from consenting patientsstimulated with a long protocol using GnRHa and gonadotropins. In Ex-periment 1, mature oocytes with a first polar body (PB) were fertilized invitro by our conventional IVF program and UFOs collected at 24 h after IVFwere activated by one of following methods: 1) no treatment (control), 2)10% ethanol, 5 min (ET), 3) 5mg/ml calcium ionophore, 5 min (CI) and 4)a single electrical DC pulse of 1.5 kV/cm, 30msec (EP). Treated UFOs weresubsequently cultured in 100ml droplets of TCM-199 medium at 37°C, 5%CO2 in air atmosphere and PN formation was evaluated by staining withHoechst 33342 at 24 h after the treatments. In Experiment 2, IOs eitherarrested at the germinal vesicle (GV), GV breakdown (GVBD) to met-aphase-I (MI) stage or classified as a mature oocyte with abnormal mor-phology at the time of retrieval were further cultured for 24–28, 8–15 and0 h, respectively. IOs were then activated by the optimal protocol ofExperiment 1 and PN formation was observed at 24 h after the treatment.Data from these experiments were analyzed by ANOVA and the leastsquare method in PLOC-GLM of SAS.

Results: In Experiment 1, there was a significant (P50.0001) treatmenteffect on PN formation in UFOs. No spontaneous activation was occurredin the control, but activation treatments induced PN formation with variousefficacy (table 1). More UFOs formed PN after ET or EP treatment thanafter CI treatment and a highest proportion (64.3%) was obtained by ET. EPwas as effective (63.5%) as ET, but fragmentation was observed in 20% ofUFOs activated by EP. Proportion of UFOs that formed presumptive hap-loid PN (2 PNs1 1 PB or 1 PN1 2 PBs) was 33.3, 0 and 28.6% after ET,CI and EP treatments, respectively. In Experiment 2, a great (P50.0362)effect of IOs’ status on PN formation was found. IOs at the GVBD-MIoocytes had higher potential to form PN than those at the GV stage or withabnormal morphology (25 vs. 77.8%).

Conclusion: The results of this study clearly demonstrated that the treat-ment of 10% ethanol for 5 min effectively induced PN formation of UFOs.IOs could form pronucleus with high efficacy by this ethanol treatment, aslong as they grew beyond the GVBD stage and maintained morphologicalnormality under the hormonal stimulation before oocyte retrieval.

* Supported by a grant from the Interdisciplinary Research Program ofthe KOSEF (1999-2-205-002-5).

THE SOCIETY OF REPRODUCTIVE SURGEONS

Wednesday, October 25, 20002:00 P.M.

O-224

Effects of Laparoscopic Ovarian Drilling on Serum Insulin and Adre-nal Steroids in Women with Polycystic Ovary Syndrome. A. Saleh, D.Morris, S. Lin Tan, T. Tulandi. Division of Reproductive Endocrinology

and Infertility, Department of Obstetrics and Gynecology, McGill Univer-sity, Montreal, Quebec, Canada.

Objectives: To evaluate insulin response to oral glucose tolerance test(OGTT) and adrenal steroid responses to ACTH stimulation in clomiphene-resistant anovulatory women with polycystic ovary syndrome (PCOS) be-fore and after laparoscopic ovarian drilling.

Design: Prospective study.Materials and Methods: 20 clomiphene-resistant anovulatory women with

PCOS underwent OGTT and ACTH tests before and after ovarian drilling.Results: Of a total 20 patients, 15 patients completed the study. 10 pa-

tients with the body mass index of.24 kg/m2 revealed evidence of insulinresistance. Positive correlation was found between serum insulin and bodyweight (r:0.6, P5.04). Insulin responses to OGTT before ovarian drilling at215, 0, 30, 60 and 120 minutes were 1926 108, 1826 102, 8636534, 10746 713 and 12006 901 pmol/L and after drilling were 162699, 1586 98, 7426 470, 9636 660 and 10856 917 pmol/L respectively(P:NS). 17a-OH progesterone responses to ACTH before and after ovariandrilling at 215, 0, 30 and 60 minutes were 2.96 1.3, 2.76 1.3, 8.96 9.4and 10.46 4.4 nmol/L and 3.06 2.2, 2.76 2.9, 6.36 2.8 and 6.66 3.0nmol/L respectively (P:NS).

Conclusions: laparoscopic ovarian drilling does not influence insulin andadrenal steroids dynamics.

Wednesday, October 25, 20002:15 P.M.

O-225

The Effect of Glucose on the Expression of Type I Collagen andTransforming Growth Factor-Beta 1 (TGF-b1) in Human PeritonealFibroblast Cells in Culture. G. Saed, W. Zhang, M. P. Diamond. De-partments of Obstetrics and Gynecology, Wayne State University School ofMedicine, Detroit, MI.

Objective: Since high glucose levels contribute to the pathogenesis of fi-brosis in organs in diabetics, we hypothesized that high glucose may alsostimulate extracellular matrix accumulation following peritoneal injury, there-by leading to adhesion development. A potential mediator for these effectsis TGF-b1, a major profibrogenic factor produced by peritoneal tissues.

Design: To test this hypothesis, we utilized the multiplex RT/PCR tech-nique to determine the effect of increasing glucose concentrations on themRNA levels of type I collagen and TGF-b1 in human peritoneal fibroblastsin culture (HPF).

Materials and Methods: Primary cultures of HPF were incubated withvarying amounts of glucose (0–5 mg/L) for 24 hours in DMEM. Total RNAwas extracted from HPF and converted to cDNA by reverse transcriptase.Multiplex RT/PCR simultaneously amplifyingb-actin with TGF-b1 or typeI collagen mRNAs in the same tube was developed in our laboratory toquantitate type I collagen and TGF-b1 mRNA levels in response to increas-ing glucose concentrations. PCR products were analyzed by agarose gelelectrophoresis and density of each ethidium bromide stained band wasmeasured by a scanning densitometer.

Results: Multiplex RT/PCR showed that there was a significant increasein the mRNA for type I collagen and TGF-b1 in response to increasingglucose concentration.

Conclusion: Increasing glucose concentration stimulated type I collagenexpression in peritoneal fibroblasts in culture. A potential mediator for thiseffect may be TGF-b1. Our results suggest that individuals with diabetesmellitus may be at risk for greater levels of postoperative adhesions.

Wednesday, October 25, 20002:30 P.M.

O-226

A Randomized Trial on the Effects of Local Installation of Bupivacaineon Postoperative Pain after Operative Laparoscopy.A. Saleh, G. Fox,A. Felemban, C. Guerra, T. Tulandi. Department of Obstetrics and Gyne-cology, and Department of Anesthesia. McGill University, Royal VictoriaHospital, Montreal, Quebec, Canada.

S84 Abstracts Vol. 74, No. 3, Suppl. 1, September 2000